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Primer combination for PCR (Polymerase Chain Reaction) identification of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination

A technology of traditional Chinese medicine formula granules and golden snake, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of DNA loss and difficult identification, and achieve the effect of overcoming the interference of excipients

Pending Publication Date: 2022-06-24
GUANGDONG YIFANG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a combination of primers for PCR identification of white snake medicinal materials, standard decoctions and traditional Chinese medicine formula granules, which has good specificity and can effectively solve the problem that DNA is difficult to identify after losing its form

Method used

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  • Primer combination for PCR (Polymerase Chain Reaction) identification of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination
  • Primer combination for PCR (Polymerase Chain Reaction) identification of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination
  • Primer combination for PCR (Polymerase Chain Reaction) identification of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Primer Design

[0067] Based on the sequence analysis of the Chinese Pharmacopoeia 2020 version of the Chinese Pharmacopoeia and common snake species NADH; Cytochrome Oxidase I; 12SrRNA; 16S rRNA; Cytochrome b, the Cytochrome Oxidase I sequences of common snake species in the GeneBank database were compared using BioEdit software. Yes, after proofreading, analyze the specific SNP site of the white snake, and import the base sequence containing the SNP site into Primer Premier 5 software for primer design.

[0068] After the sequence comparison, it was found that the specific site of the golden white snake was G and the others were A, C or T. After determining the SNP site, make the SNP site close to the 3' end of the forward primer, and adjust the primer by moving the upstream primer position. Score and GC content, and use Primer Premier 5 software to further adjust the reverse primer position, and determine the best combination through the final primer score ...

Embodiment 2

[0071] Example 2 Establishment of identification method

[0072] (1) DNA extraction and concentration adjustment

[0073]Take 0.5 g of the dried sample, grind it into powder, put it in a 2 mL centrifuge tube, add 1.5 mL of CTAB precipitation solution preheated at 56 °C and 20 μL of proteinase K, mix well, heat it in a water bath at 56 °C for 60 min, and cool it to room temperature. Centrifuge at 10,000 r / min for 5 min, discard the supernatant, add 900 μL CTAB precipitation solution and 20 μL proteinase K, and operate in the same way. Add 900 μL CTAB extract solution and 10 μL β-mercaptoethanol to the centrifuge tube in sequence, mix well, heat in a water bath at 65°C for 120 min, centrifuge, and take the supernatant after cooling to room temperature; add an equal volume of chloroform-isoamyl alcohol (24:24: 1), shake for 3 minutes, and mix well; then centrifuge at 12,000 r / min and 4°C for 10 minutes, take 750 μL of the supernatant into a new 2 mL centrifuge tube, add an equal...

Embodiment 3

[0085] Embodiment 3 Identification method verification

[0086] 14 snake samples of known species were selected (specifically shown in Table 1).

[0087] The samples were identified using the identification method established in Example 2. Among them, in the PCR system, the amount of DNA template is 50ng.

[0088] Table 1 Sample Sheet

[0089]

[0090]

[0091] Identification results such as figure 1 As shown in the figure, it can be seen from the figure that there is a 160bp band in the medicinal materials of Qianbaihuashe, the standard decoction (lyophilized powder) of Qianbaihuashe, and the traditional Chinese medicine granules of Qianbaihuashe; while other samples did not get this band. The results show that the accuracy rate of the identification method in the present invention reaches 100%.

[0092] Example 4 Methodological Validation - Annealing Temperature

[0093] (1) get the gold snake samples numbered as 1~3, 6~8 in table 1, the red chain snake numbered a...

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Abstract

The invention discloses a primer combination for PCR (Polymerase Chain Reaction) identification of bungarus multicinctus medicinal materials, standard decoction and traditional Chinese medicine formula granules, the primer combination comprises a primer I and a primer II, the primer I is a DNA (Deoxyribose Nucleic Acid) molecule with a nucleotide sequence as shown in SEQ ID NO: 1, and the primer II is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 2. The invention further discloses application of the primer combination and an identification method based on the primer combination. By implementing the method, the bungarus multicinctus medicinal material, the standard decoction and the traditional Chinese medicine formula granules can be effectively identified.

Description

technical field [0001] The invention relates to the technical field of identification of traditional Chinese medicines, in particular to a primer combination used for PCR identification of medicinal materials, standard decoction and traditional Chinese medicine formula particles, and an application and identification method thereof. Background technique [0002] There are more than 200 species of snakes in my country, of which more than 20 are common snakes, including Agkistrodonacutus, Zaocys dhumnades, Bungarus multicinctus, Bungarusfasciatus, Ptyas mucosus, round snake. Daboia russelii, Naja naja, Lycodon rufozonatus, Elaphe carinata, Ptyas korros, Gloydiusbrevicaudus, Elaphe taeniura, Elaphe moellendorffi, etc. Has clinical efficacy. Due to the similar shapes of various snakes, it is difficult to identify medicinal materials, especially after the internal organs are cut off during processing, they must be dried and blackened. Standard decoction, after further processing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6806C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/6806C12Q1/686C12Q2527/125C12Q2521/537C12Q2565/125
Inventor 李国卫魏梅罗宇琴陈向东邓淙友范耀耀宋叶黄上书
Owner GUANGDONG YIFANG PHARMA
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